The integrins are a class of heterodimeric integral membrane proteins, one or more of which are expressed by most cell types (Hynes (1992) Cell 69:11–25). Some 16 homologous alpha subunits and 8 homologous beta subunits associate in various combinations to yield an extensive family of receptors. Each integrin heterodimer has a large extracellular domain that mediates binding to specific ligands. These ligands may include plasma proteins, proteins expressed on the surface of adjacent cells, or components of the extracellular matrix. Several of the integrins show affinity for more than one ligand and many have overlapping specificities (Hynes (1992) Cell 69:11–25). Both the α and β subunits contribute to a small intracellular domain that contacts components of the actin cytoskeleton, thus forming a physical link between proteins outside and inside the cell. Integrins play an important role in cellular adhesion and migration, and these properties are controlled by the cell, in part, by modulation of integrin affinity for its ligands (so-called “inside-out” signaling). Conversely, the presence or absence of integrin ligation provides specific information about the cellular microenvironment, and in many instances integrins serve as a conduit for signal transduction. Ligand binding by an integrin may promote its incorporation into focal adhesions, the assembly of cytoskeletal and intracellular signaling molecules into supra-molecular complexes, and the initiation of a cascade of downstream signaling events including protein phosphorylation, calcium release, and an increase in intracellular pH (reviewed by Schwartz et al. (1995) Ann. Rev. Cell Dev. Biol. 11:549–99). Such “outside-in” signaling ties into pathways controlling cell proliferation, migration and apoptosis (Stromblad et al. (1996) J. Clin. Invest. 98:426–33; Eliceiri et al. (1998) J. Cell. Biol. 140:1255–63). Integrins have been shown to play a role in such diverse physiological settings as embryonic development, wound healing, angiogenesis, clot formation, leukocyte extravasation, bone resorption and tumor metastasis.
The β3-containing integrins are among the best studied of the receptor superfamily. The β3 subunit forms heterodimers with either αv (αvβ3) or αIIb (αIIbβ3). While these integrins show substantial overlap in ligand specificity, they play very different roles in normal physiology and in disease.
αvβ3 is expressed by activated endothelial cells, smooth muscle cells, osteoclasts, and, at a very low level, by platelets. It is also expressed by a variety of tumor cell types. The integrin binds to a number of plasma proteins or proteins of the extracellular matrix, many of which are associated with sites of inflammation or wound healing (Albelda (1991) Am. J. Resp. Cell Mol. Biol. 4:195–203). These include vitronectin, fibronectin, osteopontin, von Willebrand factor, thrombospondin, fibrinogen, and denatured collagen Type I (Hynes (1992) Cell 69:11–25). Each of these proteins share a common sequence motif, arginine-glycine-aspartic acid (RGD), that forms the core of the integrin binding site.
αvβ3 has been most intensely studied in the context of new blood vessel formation (angiogenesis) where it mediates the adhesion and migration of endothelial cells through the extracellular matrix. Angiogenesis in adults is normally associated with the cyclical development of the corpus luteum and endometrium and with the formation of granulation tissue during wound repair. In the latter case, microvascular endothelial cells form vascular sprouts that penetrate into the temporary matrix within a wound. These cells transiently express αvβ3 and inhibition of the ligand binding function of the integrin temporarily inhibits the formation of granulation tissue (Clark et al. (1996) Am. J. Pathol. 148:1407–21). In cytokine-stimulated or unstimulated angiogenesis on the chick chorioallantoic membrane, blockade of αvβ3 with a heterodimer-specific antibody prevents new vessel formation without affecting the pre-existing vasculature (Brooks et al. (1994) Science 264:569–71). Furthermore, the loss of adhesive contacts by endothelial cells activated for angiogenesis induces a phenotype characteristic of apoptotic cells (Brooks et al. (1994) Cell 79:1157–64); that is, ligand binding by αvβ3 appears to transmit a survival signal to the cell. Thus, adhesion and/or signaling mediated by αvβ3 is essential for the formation of new blood vessels.
Solid tumors are unable to grow to significant size without an independent blood supply. It is currently hypothesized that the acquisition of an angiogenic phenotype is one of the limiting steps in the growth of primary tumors and of tumors at secondary sites (Folkman (1995) Nat. Med. 1:27–31). In addition, while the vasculature that penetrates a tumor mass provides a source of oxygen and nutrients, it also serves as a conduit for metastatic cells to leave the primary tumor and migrate throughout the body. Thus, inhibition of angiogenesis may limit both the growth and metastasis of cancerous lesions. In experimental settings of tumor-induced angiogenesis, inhibition of ligand-binding by endothelial αvβ3 prevented the formation of new blood vessels (Brooks et al. (1994) Cell 79:1157–64; Brooks et al. (1995) J. Clin. Invest. 96:1815–22), and inhibitors of αvβ3 were shown to reduce the growth of experimental tumors in vivo (Brooks et al. (1995) J. Clin. Invest. 96:1815–22; Carron et al. (1998) Canc. Res. 58:1930–5).
αvβ3 is not only expressed by the microvasculature within tumors, but in some cases, is also found on the surface of tumor cells themselves. In particular, expression of αvβ3 integrin has been detected in tissue sections from tumors of melanocytic and astroglial origin (Albelda et al. (1990) Canc. Res. 50:6757–64; Gladson and Cheresh (1991) J. Clin. Invest. 88:1924–32), and the level of integrin expression has been correlated with the stage or metastatic potential of the tumor (Albelda et al. (1990) Canc. Res. 50:6757–64; Gladson et al. (1996) Am. J. Pathol. 148:1423–34; Hieken et al. (1996) J. Surg. Res. 63:169–73). Furthermore, melanoma cells grown in vitro in a three-dimensional matrix of denatured collagen undergo apoptosis upon αvβ3 blockade.
Data such as these have driven an interest in inhibitors of αvβ3 for the treatment of cancer. At present, two such inhibitors are in or near clinical trial: Vitaxin is a chimeric Fab fragment derived from the αvβ3-specific monoclonal antibody, LM609 (Wu et al. (1998) Proc. Nat. Acad. Sci. 95:6037–42). A phase I trial in late-stage cancer patients has been completed and no significant treatment-associated toxicities were observed (Gutheil et al. (1998) Am. Soc. Clin. Onc.). EMD121974 is a cyclic pentapeptide inhibitor of αvβ3. A Phase I study of this compound in Kaposi's sarcoma, brain tumors and solid tumors is scheduled to begin in 1999.
Angiogenesis (and αvβ3) are implicated in the pathology of several other diseases, including psoriasis (Creamer et al. (1995) Am. J. Pathol. 147:1661–7), rheumatoid arthritis (Walsh et al. (1998) Am. J. Pathol. 152:691–702; Storgard et al. (1999) J. Clin. Invest. 103:47–54), endometriosis (Healy et al. (1998) Hum. Reprod. Update 4:736–40), and several proliferative diseases of the eye (Casaroli Marano et al. (1995) Exp. Eye Res. 60:5–17; Friedlander et al. (1996) Proc. Nat. Acad. Sci. 93:9764–9; Hammes et al. (1996) Nat. Med. 2:529–33). Inhibition of integrin ligand binding in each of these contexts may provide significant therapeutic benefit.
Atheromatous plaque and restenosis following angioplasty are pathologies characterized by thickening of the intima, the innermost layer of the arterial wall. The proliferation and/or migration of smooth muscle cells into the neointima with concomitant deposition of fibrous extracellular proteins contributes to vessel wall thickening and subsequent vessel occlusion. Platelets may also contribute to the development of restenotic lesions through adhesion to endothelial cells and the release of growth factors and cytokines that stimulate the underlying smooth muscle cell layer (Le Breton et al. (1996) J. Am. Coll. Cardiol. 28:1643–51). αvβ3 integrin is expressed on arterial smooth muscle cells (Hoshiga et al. (1995) Circ. Res. 77:1129–35) and mediates their migration on vitronectin and osteopontin (Brown et al. (1994) Cardiovasc. Res. 28:1815–20; Jones et al. (1996) Proc. Nat. Acad. Sci. 93:2482–7; Liaw et al. (1995) J. Clin. Invest. 95:713–24; Panda et al. (1997) Proc. Nat. Acad. Sci. 94:9308–13), both matrix proteins that are associated with atheroschlerotic tissues in vivo (Brown et al. (1994) Cardiovasc. Res. 28:1815–20; Giachelli et al. (1995) Ann. N. Y. Acad. Sci. 760:109–26; Panda et al. (1997) Proc. Nat. Acad. Sci. 94:9308–13). In addition, αvβ3 expression on endothelial cells, and to a much lesser extent on platelets, is responsible for at least part of the adhesive interaction between these cell types (Le Breton et al. (1996) J. Am. Coll. Cardiol. 28:1643–51; Gawaz et al. (1997) Circulation 96:1809–18). αvβ3 blockade with RGD-containing peptides or a monoclonal antibody was found to limit neointimal hyperplasia in several animal models of restenosis following arterial injury (Choi et al. (1994) J. Vasc. Surg. 19:125–34; Srivatsa et al. (1997) Cardiovasc. Res. 36:408–28; Slepian et al. (1998) Circulation 97:1818–27; Coleman et al. (1999) Circ. Res. 84:1268–76). Furthermore, treatment of patients undergoing percutaneous coronary intervention with an anti-β3 antibody (Reopro/abciximab/c7E3), which blocks both the platelet fibrinogen receptor, αIIbβ3, and αvβ3, provided long term reduction in the rates of death or myocardial infarction and in the rate of reocclusion of the artery (Lefkovits et al. (1996) Am. J. Cardiol. 77:1045–51), an effect that may be mediated through inhibition of αvβ3 ligation. The observation that αvβ3 is expressed by microvascular smooth muscle cells after experimentally-induced focal cerebral ischemia (Okada et al. (1996) Am. J. Pathol. 149:37–44) suggests that this integrin may also play some role in the development of ischemia/reperfusion injury in stroke.
Finally, αvβ3 mediates the attachment of osteoclasts to matrix proteins, particularly osteopontin, on the surface of bone. Osteoclasts are responsible for the resorption of bone in normal physiology as well as in pathological conditions such as osteoporosis. A monoclonal antibody specific for αvβ3 inhibited the binding and resorption of bone particles by osteoclasts in vitro (Ross et al. (1993) J. Biol. Chem. 268:9901–7). Furthermore, an RGD-containing protein, echistatin, was shown to block parathyroid-stimulated bone resorption in an animal model, as monitored by serum calcium levels (Fisher et al. (1993) Endocrin. 132:1411–3). Inhibitors of αvβ3 integrin are thus considered of potential utility in treating debilitating bone loss such as occurs in osteoporosis.
αIIbβ3 (also referred to as GPIIbIIIa) is the major integrin on the surface of platelets where it mediates the adhesion of activated platelets to the plasma protein fibrinogen (Nachman and Leung (1982) J. Clin. Invest. 69:263–9; Shattil et al. (1985) J. Biol. Chem. 260:11107–14). During clot formation, fibrinogen dimers cross-link platelets to one another through the integrin receptor. αIIbβ3 also binds to several other plasma and cell matrix proteins, including von Willebrand factor, vitronectin, and fibronectin (Faull and Ginsberg (1996) J. Am. Soc. Nephrol. 7:1091–7).
Clot formation is a tightly regulated process that balances the need for rapid response to vascular injury with the risk of aberrant occlusion of critical vessels. The αIIbβ3 heterodimer is constitutively expressed on the surface of resting platelets at approximately 80,000 copies per cell (Wagner et al. (1996) Blood 88:907–14); however, the affinity of the integrin for fibrinogen is very low on these cells. Activation of platelets by ADP, epinephrine, collagen or thrombin leads to a dramatic enhancement in integrin ligand binding activity (Bennett and Vilaire (1979) J. Clin. Invest. 64:1393–401; Marguerie et al. (1979) J. Biol. Chem. 254:5357–63), probably accomplished through a conformational change in the receptor (Shattil et al. (1985) J. Biol. Chem. 260:11107–14; O'Toole et al. (1990) Cell Reg. 1:883–93; Du et al. (1993) J. Biol. Chem. 268:23087–92). In this prototypic example of “inside-out” control of integrin function, cross-linking of platelets through the αIIbβ3-fibrinogen interaction is confined to local sites of platelet activation.
Inhibitors of αIIbβ3 ligand binding have been primarily explored in the context of cardiovascular disease (Chong (1998) Am. J. Health Syst. Pharm. 55:2363–86; Topol et al. (1999) Lancet 353:227–31), but may have application in any of a number of indications where thrombus formation is suspected or is likely. Three αIIbβ3 inhibitors have been approved for use in patients experiencing acute coronary syndrome and/or in patients who are undergoing percutaneous coronary intervention. Reopro (Centocor/Eli Lilly) is a humanized murine monoclonal antibody Fab fragment with specificity for the β3 chain of αIIbβ3. Integrilin (COR Therapeutics) is a cyclic heptapeptide based on the integrin binding site of barbourin, an αIIbβ3 inhibitory protein derived from snake venom. Aggrastat (Merck & Co.) is a non-peptide small molecule antagonist of the integrin. Unlike the small molecule inhibitors, Reopro cross-reacts with αvβ3, a fact which may account for the greater reduction in long-term rates of death and non-fatal myocardial infarction associated with its use (see above). A significant effort is underway to identify new inhibitors of the platelet integrin with characteristics not found in the cohort of approved drugs. Specifically, compounds with specificity for the active, ligand-binding conformation of αIIbβ3 may reduce the risk of bleeding complications associated with the existing anti-clotting therapies. Orally available compounds would be particularly useful for longer term therapy of patients at risk for recurrent myocardial infarction or unstable angina.
Given the role of integrins in the various disease states described above, it would be desirable to have high specificity inhibitors of particular integrins. The present invention provides such agents.
The dogma for many years was that nucleic acids had primarily an informational role. Through a method known as Systematic Evolution of Ligands by EXponential enrichment, termed the SELEX process, it has become clear that nucleic acids have three dimensional structural diversity not unlike proteins. The SELEX process is a method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules and is described in U.S. patent application Ser. No. 07/536,428, filed Jun. 11, 1990, entitled “Systematic Evolution of Ligands by EXponential Enrichment,” now abandoned, U.S. Pat. No. 5,475,096, entitled “Nucleic Acid Ligands” and U.S. Pat. No. 5,270,163 (see also WO 91/19813), entitled “Methods for Identifying Nucleic Acid Ligands,” each of which is specifically incorporated by reference herein in its entirety. Each of these applications, collectively referred to herein as the SELEX Patent Applications, describes a fundamentally novel method for making a nucleic acid ligand to any desired target molecule. The SELEX process provides a class of products which are referred to as nucleic acid ligands or aptamers, each having a unique sequence, and which has the property of binding specifically to a desired target compound or molecule. Each SELEX-identified nucleic acid ligand is a specific ligand of a given target compound or molecule. The SELEX process is based on the unique insight that nucleic acids have sufficient capacity for forming a variety of two- and three-dimensional structures and sufficient chemical versatility available within their monomers to act as ligands (form specific binding pairs) with virtually any chemical compound, whether monomeric or polymeric. Molecules of any size or composition can serve as targets. The SELEX method applied to the application of high affinity binding involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific high affinity nucleic acid ligands to the target molecule.
It has been recognized by the present inventors that the SELEX method demonstrates that nucleic acids as chemical compounds can form a wide array of shapes, sizes and configurations, and are capable of a far broader repertoire of binding and other functions than those displayed by nucleic acids in biological systems.
The basic SELEX method has been modified to achieve a number of specific objectives. For example, U.S. patent application Ser. No. 07/960,093, filed Oct. 14, 1992, now abandoned, and U.S. Pat. No. 5,707,796, both entitled “Method for Selecting Nucleic Acids on the Basis of Structure,” describe the use of the SELEX process in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA. U.S. patent application Ser. No. 08/123,935, filed Sep. 17, 1993, entitled “Photoselection of Nucleic Acid Ligands,”, now abandoned, U.S. Pat. No. 5,763,177, entitled “Systematic Evolution of Ligands by Exponential Enrichment: Photoselection of Nucleic Acid Ligands and Solution SELEX” and U.S. patent application Ser. No. 09/093,293, filed Jun. 8, 1998, entitled “Systematic Evolution of Ligands by Exponential Enrichment: Photoselection of Nucleic Acid Ligands and Solution SELEX,” describe a SELEX based method for selecting nucleic acid ligands containing photoreactive groups capable of binding and/or photocrosslinking to and/or photoinactivating a target molecule. U.S. Pat. No. 5,580,737, entitled “High-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeine,” describes a method for identifying highly specific nucleic acid ligands able to discriminate between closely related molecules, which can be non-peptidic, termed Counter-SELEX. U.S. Pat. No. 5,567,588, entitled “Systematic Evolution of Ligands by EXponential Enrichment: Solution SELEX,” describes a SELEX-based method which achieves highly efficient partitioning between oligonucleotides having high and low affinity for a target molecule.
The SELEX method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX process-identified nucleic acid ligands containing modified nucleotides are described in U.S. Pat. No. 5,660,985, entitled “High Affinity Nucleic Acid Ligands Containing Modified Nucleotides,” that describes oligonucleotides containing nucleotide derivatives chemically modified at the 5- and 2′-positions of pyrimidines. U.S. Pat. No. 5,580,737, supra, describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2′-amino (2′-NH2), 2′-fluoro (2′-F), and/or 2′-O-methyl (2′-OMe). U.S. patent application Ser. No. 08/264,029, filed Jun. 22, 1994, entitled “Novel Method of Preparation of Known and Novel 2′ Modified Nucleosides by Intramolecular Nucleophilic Displacement,” now abandoned, describes oligonucleotides containing various 2′-modified pyrimidines.
The SELEX method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in U.S. Pat. No. 5,637,459, entitled “Systematic Evolution of Ligands by EXponential Enrichment: Chimeric SELEX,” and U.S. Pat. No. 5,683,867, entitled “Systematic Evolution of Ligands by EXponential Enrichment: Blended SELEX,” respectively. These applications allow the combination of the broad array of shapes and other properties, and the efficient amplification and replication properties, of oligonucleotides with the desirable properties of other molecules.
The SELEX method further encompasses combining selected nucleic acid ligands with lipophilic compounds or non-immunogenic, high molecular weight compounds in a diagnostic or therapeutic complex as described in U.S. patent application Ser. No. 08/434,465, filed May 4, 1995, entitled “Nucleic Acid Ligand Complexes”. Each of the above described patent applications which describe modifications of the basic SELEX procedure are specifically incorporated by reference herein in their entirety.
It is an object of the present invention to provide methods that can be used to identify nucleic acid ligands that bind with high specificity and affinity to particular integrins.
It is a further object of the present invention to obtain nucleic acid ligands to particular integrins that inhibit the ability of that integrin to bind its cognate ligand.
It is a further object of the present invention to obtain integrin inhibiting pharmaceutical compositions for controlling thrombosis, tumor angiogenesis, tumor cell migration, proliferative ocular diseases, rheumatoid arthritis, psoriasis, osteoporosis, and restenosis.
It is yet a further object of the invention to obtain imaging agents for the non-invasive detection of deep vein or arterial thrombi.